PDBsum entry 3tk3

Oxidoreductase

PDB id: 3tk3

Contents

Protein chains

465 a.a.

Ligands

HEM-CPZ ×4

Waters ×290

PDB id:

3tk3

Name:

Oxidoreductase

Title:

Cytochrome p450 2b4 mutant l437a in complex with 4-(4-chlorophenyl) imidazole

Structure:

Cytochrome p450 2b4. Chain: a, b, c, d. Synonym: cypiib4, cytochrome p450 isozyme 2, cytochrome p450 lm2, cytochrome p450 type b0, cytochrome p450 type b1. Engineered: yes. Mutation: yes

Source:

Oryctolagus cuniculus. European rabbit,japanese white rabbit,domestic rabbit,rabbits. Organism_taxid: 9986. Gene: cyp2b4. Expressed in: escherichia coli. Expression_system_taxid: 562.

Resolution:

2.80Å R-factor: 0.195 R-free: 0.241

Authors:

S.C.Gay,H.H.Jang,P.R.Wilderman,Q.Zhang,C.D.Stout,J.R.Halpert

Key ref:

P.R.Wilderman et al. (2012). Investigation by site-directed mutagenesis of the role of cytochrome P450 2B4 non-active-site residues in protein-ligand interactions based on crystal structures of the ligand-bound enzyme. Febs J, 279, 1607-1620. PubMed id: 22051155

Date:

25-Aug-11 Release date: 16-Nov-11

Protein chains

P00178  (CP2B4_RABIT) -  Cytochrome P450 2B4 from Oryctolagus cuniculus

Seq: 491 a.a.

Struc: 465 a.a.*

* PDB and UniProt seqs differ at 4 residue positions (black crosses)

Enzyme reactions

• Enzyme class: E.C.1.14.14.1 - unspecific monooxygenase.
• Reaction: an organic molecule + reduced [NADPH--hemoprotein reductase] + O2 = an alcohol + oxidized [NADPH--hemoprotein reductase] + H2O + H⁺
• Cofactor: Heme-thiolate

Molecule diagrams generated from .mol files obtained from the KEGG ftp site

Added reference

Febs J 279:1607-1620 (2012)

PubMed id: 22051155

Investigation by site-directed mutagenesis of the role of cytochrome P450 2B4 non-active-site residues in protein-ligand interactions based on crystal structures of the ligand-bound enzyme.

P.R.Wilderman, S.C.Gay, H.H.Jang, Q.Zhang, C.D.Stout, J.R.Halpert.

ABSTRACT

Residues located outside the active site of cytochromes P450 2B have exhibited importance in ligand binding, structural stability and drug metabolism. However, contributions of non-active-site residues to the plasticity of these enzymes are not known. Thus, a systematic investigation was undertaken of unique residue-residue interactions found in crystal structures of P450 2B4 in complex with 4-(4-chlorophenyl)imidazole (4-CPI), a closed conformation, or in complex with bifonazole, an expanded conformation. Nineteen mutants distributed over 11 sites were constructed, expressed in Escherichia coli and purified. Most mutants showed significantly decreased expression, especially in the case of interactions found in the 4-CPI structure. Six mutants (H172A, H172F, H172Q, L437A, E474D and E474Q) were chosen for detailed functional analysis. Among these, the K(s) of H172F for bifonazole was ∼ 20 times higher than for wild-type 2B4, and the K(s) of L437A for 4-CPI was ∼ 50 times higher than for wild-type, leading to significantly altered inhibitor selectivity. Enzyme function was tested with the substrates 7-ethoxy-4-(trifluoromethyl)coumarin, 7-methoxy-4-(trifluoromethyl)coumarin and 7-benzyloxyresorufin (7-BR). H172F was inactive with all three substrates, and L437A did not turn over 7-BR. Furthermore, H172A, H172Q, E474D and E474Q showed large changes in k(cat)/K(M) for each of the three substrates, in some cases up to 50-fold. Concurrent molecular dynamics simulations yielded distances between some of the residues in these putative interaction pairs that are not consistent with contact. The results indicate that small changes in the protein scaffold lead to large differences in solution behavior and enzyme function.